JPH09224672A - Dna coding new dna-connected protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a DNA as a cysteine-rich DNA connecting protein gene from a gene specifically generating after treating with a jasmonic acid. SOLUTION: This DNA has an amino acid sequence of the formula and codes a DNA-connecting protein containable substitution, deletion, insertion, addition or transition of more than one amino acid as far as not substantially injuring a DNA connecting activity, and is, e.g., SJIP-2 as one of the gene obtained by a jasmonic acid treatment of a culturing cell of a soybean. The DNA connecting protein is considered as a factor controlling manifestation of a gene induced by jasmonic acid and is useful for producing a plant strong against a pathogenic fungus, producing a useful secondary metabolite and suppressing generation of a toxic secondary metabolite, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA-binding protein gene, and more particularly to a cysteine-rich DNA-binding protein gene specifically induced by jasmonic acid and use thereof.

[0002]

2. Description of the Related Art Jasmonic acid is a substance isolated as one of the aroma components of jasmine oil obtained from jasmine flowers, and includes tuber formation of potatoes, fruit ripening, resistance reaction to plant pathogens, and plant It is known to have physiological actions such as growth inhibition, promotion of leaf senescence, and control of stomatal opening and closing. In addition, many proteins whose expression is induced by jasmonic acid (Jsmonate induced protein; JI
P) has been found and is also known as a genetic information substance, and research on gene expression regulation using jasmonic acid is also underway.

Among the gene products induced by jasmonic acid as described above, there are proteinase inhibitors (Proc. Natl. Acad. Sci. USA 87, 7713 (1990)) and ribosome inactivating proteins (Proc. Natl. Acad. Sci.
USA 91, 7012 (1994)), plant storage protein (vegetati
ve storage protein (Plant Sci. 62, 45 (1989)), lipoxygenase (Plant Cell Physiol. 34, 1063 (199
3)), proteins related to defense mechanism against attack of pathogenic bacteria (Annu. Rev. Plant Physiol. Plant Mol. Biol. 44,
569 (1993)) and other secondary metabolites of plants (Proc. Nat.
l. Acad. Sci. USA92, 12505 (1995)) is known to exist.

Therefore, if the expression of these jasmonic acid-induced genes can be controlled, it is possible to accumulate a protective substance against pathogenic bacteria and increase or decrease the amount of secondary metabolite synthesis. It is considered to be.

By the way, it is considered that a DNA binding protein that binds to a promoter region is involved in the regulation of gene expression. Therefore, as one method for controlling the expression of the gene, it is considered to manipulate such a DNA binding protein gene. That is, if the expression of the DNA binding protein gene can be freely controlled, the expression of various genes regulated by it can be controlled, and various phenomena can be caused.

[0006] Therefore, if the gene of the DNA binding protein whose expression is induced by jasmonic acid is isolated, it becomes possible to control the expression of the gene induced by jasmonic acid by using it, and the pathogenic bacterium It is considered possible to produce highly resistant plants or plants with increased / decreased amounts of secondary metabolites.

However, the DNA binding protein induced by jasmonic acid and its gene have not been obtained and known, and an attempt to utilize such a gene to control the amount of secondary metabolites by jasmonic acid. Was of course not done.

[0008]

SUMMARY OF THE INVENTION The present invention has been made from the above point of view, and it is an object of the present invention to analyze the expression mechanism of genes by jasmonic acid and to control the expression of genes using jasmonic acid.

[0009]

[Means for Solving the Problems] The present inventor has conducted research on a gene that specifically appears after treatment with jasmonic acid, and succeeded in isolating one of such genes. Was found to be a DNA binding protein gene.

Specifically, soybean culture cells were treated with jasmonic acid, a plurality of cDNA clones induced by jasmonic acid were isolated by the differential display method, and the nucleotide sequences were determined. And one of these
One is Cystein-rich DNA-binding protein that binds to the X-box conserved in the promoter region of human MHC class II gene.
tein; NF-X1) has a homology,
The present invention has been completed.

That is, the present invention has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and has one or more amino acid substitutions, deletions, insertions, additions or transfers unless the DNA binding activity is substantially impaired. It may be a DNA-binding protein and a DNA encoding the same. Here, the "DNA binding activity" is specifically the property of the DNA binding protein of the present invention binding to the promoter region of a gene and participating in the expression control of the gene.

Specifically, as the above DNA, for example, in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, nucleotide number 45
DNA having a base sequence represented by 0 to 3480 can be mentioned. The DNA of the present invention is not limited to the above-mentioned nucleotide sequences, and includes DNAs having different nucleotide sequences encoding the same amino acid sequence due to the degeneracy of the genetic code.

The present invention also provides a DNA having at least a part of a base sequence (antisense sequence) complementary to the base sequence (sense sequence) shown in SEQ ID NO: 1. Furthermore, the present invention provides a plant transformed with a DNA having the above sense sequence or antisense sequence.

The DNA binding protein of the present invention is NF-
It has homology with the X1 protein. The NF-X1 protein is a human MHC (major histocompatibility c
omplex, MHC: major histocompatibility complex) is a protein that binds to X-box, which is conserved in the promoter region of class II genes, and has been reported to function as a repressor that controls the genes ( J. Exp.
Med. 180, 1763 (1994)). The above MHC class II gene was initially identified as a gene that controls the success or failure of allograft engraftment, and has subsequently been proved to be a gene that determines the difference in immune responsiveness between individuals and systems. It has been shown to play an important role in the immune system of animals.

The expression of the gene for the DNA-binding protein of the present invention was confirmed to be specifically induced by jasmonic acid when the expression analysis was carried out using cDNA as a probe.

[0016] Jasmonic acid is involved in various plant reactions such as biosynthesis of secondary metabolites, and has a function as an information transmitter controlling the expression of many genes. The DNA binding protein of the present invention is considered to be a factor that regulates the expression of these jasmonic acid-induced genes. Therefore, it is considered possible to control the expression of those genes, accumulate a protective substance against pathogenic bacteria, and increase or decrease the amount of secondary metabolite synthesis. In particular,
The DNA of the present invention can be expressed in plants, or the DN of the present invention can be expressed.
It is expected that by expressing the antisense gene of A in plants, it is possible to produce plants resistant to pathogenic bacteria, to produce useful secondary metabolites, and to suppress the production of toxic secondary metabolites. To be done.

According to the present invention, a DNA encoding a DNA binding protein induced by jasmonic acid was obtained. Therefore, a hybridization method using this sequence or an oligonucleotide prepared based on this sequence as a probe, or the above oligo A DNA-binding protein gene can be isolated from plant chromosomal DNA by the PCR (polymerase chain reaction) method using nucleotides as primers. Since the expression of this gene itself is induced by jasmonic acid, not only the gene product but also its promoter can be used for regulating gene expression in the plant body.

[0018]

Next, an embodiment of the present invention will be described. Since the base sequence and the amino acid sequence encoded thereby by the DNA of the present invention have been clarified, one set of oligonucleotides synthesized based on the amino acid sequence or prepared based on the base sequence is used as a primer. By a PCR method or a hybridization method using an oligonucleotide prepared based on the nucleotide sequence as a probe, chromosomal DNA, mRNA or c of a plant
It can be isolated from DNA. When the gene for the DNA-binding protein of the present invention is obtained from the chromosomal DNA by amplification by the PCR method, it is highly likely that an intron is contained, but one or more such introns are contained inside. Those containing are also included in the DNA of the present invention.

Of the above methods, the PCR method is preferred, but when the present invention is completed, the DNA of the present invention is subjected to the differential display method (Science 257, 967).
(1992)).

The DNA of the present invention can be obtained by isolating a cDNA specifically induced by jasmonic acid from plant cells such as soybean culture cells. Specifically, cDNA is obtained from only jasmonic acid-treated cultured cells by extracting RNA from the jasmonic acid-treated cultured cells and untreated cultured cells and performing the differential display method (Science 257, 967 (1992)). Is isolated. That is, cDNA was synthesized from RNA prepared from jasmonic acid-treated cells and untreated cultured cells, each was subjected to gel electrophoresis, and it was found only in cDNA derived from jasmonic acid-treated cells. By extracting the band in which no visible band is observed from the gel, cDNA derived from the gene induced by jasmonic acid can be obtained.

When the nucleotide sequence of the cDNA obtained as described above is determined and it is recognized that it does not include the entire coding region, colony hybridization or plaque hybridization using the obtained cDNA as a probe Hybridization positive clones may be screened from a plant cell-derived cDNA library.

The cDNA library is prepared from plant tissues.
It can be prepared by extracting RNA, synthesizing cDNA using reverse transcriptase, inserting the double-stranded product by polymerase reaction into a vector, and transforming into Escherichia coli or the like. Since cDNA cloning kits are commercially available, these may be used.

The vector used for preparing the library is
Many types are commercially available and these can be used. The methods necessary for general gene recombination such as DNA cleavage, ligation, transformation, gene nucleotide sequence determination, and hybridization are described in the instructions attached to commercially available enzymes used for each operation, and Molecular cloning. (Maniatis T.
et al. Cold Spring Harbor Laboratory Press).

When a hybridization positive clone is obtained as described above, its nucleotide sequence is determined. The nucleotide sequence is determined by the Maxam-Gilbert method or the dideoxy method. The nucleotide sequence can be determined by the dideoxy method using a commercially available kit, and an autosequencer for automatically performing the sequence determination may be used.

DNA of the present invention obtained as described above
The nucleotide sequence of is shown in SEQ ID NO: 1. The amino acid sequence deduced from this base sequence is shown in SEQ ID NO: 2. A database analysis of this amino acid sequence revealed that the amino acid sequences 1 to 771 were cysteine-rich DNAs that bind to X-box conserved in the promoter region of human MHC class II gene.
Binding protein (Cystein-rich DNA-binding protei
It was found to have a homology of 39.7% with the amino acid sequence of n (NF-X1)) from 343 to 1087, and it was shown to be a DNA binding protein. still,
In SEQ ID NO: 1, ATG having base numbers 450 to 452
Although the coding region is shown as a start codon, this means that this codon is most likely the start codon, and does not completely deny that the coding region continues upstream. . However, from the homology with NF-X1, it is considered that the amino acid sequence shown in SEQ ID NO: 2 is sufficient to exhibit the activity as a DNA binding protein.

The gene for the above DNA-binding protein is S
It was named JIP-2. Northern analysis was performed to examine the expression pattern of this gene. That is, RNA was extracted from cells treated with jasmonic acid and untreated cells, agarose gel electrophoresis was performed, and RNA was transferred from the gel to a membrane. When hybridization was carried out using the previously obtained cDNA as a probe, SJIP-2
No expression was observed in untreated cells, and it was revealed that it was specifically expressed in cells treated with jasmonic acid. Therefore, it was revealed that SJIP-2 is a gene of a DNA binding protein specifically induced by jasmonic acid.

According to the present invention, a cDNA of a DNA-binding protein specifically induced by jasmonic acid was obtained. It was prepared from a chromosomal DNA library by hybridization using this sequence or based on this cDNA sequence. From the chromosomal DNA to SJIP-2 by the PCR method using the oligonucleotide as a primer.
Can be isolated.

All or part of the DNA or SJIP-2 of the present invention is linked to a plant-expressible promoter such as a promoter derived from cauliflower mosaic virus,
A plant that highly expresses a DNA-binding protein can be prepared by introducing a recombinant DNA containing this into a plant and transforming it. In addition, the DNA or S of the present invention
All or part of JIP-2 bound to a promoter in the reverse direction was introduced into a plant to obtain so-called antisense R.
By expressing NA, the translation of the DNA binding protein mRNA can be inhibited and the expression level can be suppressed.

Plant transformation is carried out by the particle gun method,
It can be carried out by introducing DNA into protoplasts by electroporation (electroporation method) or a method using Ti plasmid of Agrobacterium.

The selection of clones into which the SJIP-2 sense or antisense gene has been introduced can be carried out by collecting callus or plant cells obtained from the transformed cells and confirming them by a method such as Southern hybridization. Good. After regeneration, it is preferable to extract RNA from the leaves of the plant and examine the expression of the DNA-binding protein gene by Northern analysis or the like, while confirming that SJIP-2 has been introduced by Southern analysis or the like.

[0031]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. <1> cDN specifically expressed by jasmonic acid treatment
Isolation by differential display method of A. Soybean cultured cells (SB-P cells, provided by Professor Jack M. Widholm. See Plant Physiol. 72, 426 (1983)).
20 μM jasmonic acid methyl ester and 100
After treatment with μM cycloheximide for 30 minutes to generate jasmonic acid-induced mRNA while inhibiting de novo protein synthesis, guanidine was obtained from jasmonic acid-treated cells and untreated cells as shown below. RNA was extracted by the thiocyanate / CsCl method.

Approximately 1 g of each cultured cell was ground in liquid nitrogen, placed in 14 ml of 4M guanidine thiocyanate, 117 mM mercaptoethanol, 25 mM sodium acetate solution, and ground completely with a Polytron homogenizer. After centrifuging this at 13,000 xg for 25 minutes, the supernatant was layered on 1.5 ml of a 5.7 M CsCl solution in a centrifuge tube. This is 18
After centrifugation at 2,000 xg for 20 hours, the supernatant was removed.
The precipitate was dissolved in TE buffer to obtain total RNA.

Next, using each RNA, Gne
The differential display method was performed using the RNAmap kit from Hunter. PCR primers have 4 kinds of oligo dT primers shown in SEQ ID NOS: 3 to 6 (V indicates that G, A, and C are mixed in each sequence) and 20 kinds of 10-mer random sequences. The primers were used in combination. All of these primers are included in the kit.

Using 0.2 μg of the above RNA as a template,
Reverse transcriptase (MMLV (Moloney Murine Leukemia Virus) re
verse transcriptase) was used to synthesize cDNA, then the above primers were added, and the temperature was 94 ° C for 30 seconds and 40 ° C.
An amplification reaction consisting of 2 minutes and 72 ° C. for 30 seconds was performed for 40 cycles. [ ³⁵ S] -dATP was added to the reaction solution to incorporate [ ³⁵ S] -dATP into the amplification product.

Each reaction product was subjected to electrophoresis using a sequencing gel (gel for determining a nucleotide sequence), an autoradiogram was taken, and then a band that appeared specifically in the jasmonic acid treatment was examined. RNA derived from untreated cells amplified from RNA derived from jasmonic acid-treated cells
DNA bands that were not amplified were excised from the gel and boiled in TE buffer to
A was extracted. Based on the obtained DNA, PCR was carried out using the same primers as in the first reaction to amplify the DNA. The amplified DNA was transformed into pCRII vector (Invitroge
(manufactured by company n) and used for the subsequent analysis.

<2> Isolation of long-chain cDNA Cultured soybean cells were treated with 50 μM jasmonic acid for 1 hour, and total R was measured by the guanidine thiocyanate / CsCl method.
NA was extracted. Poly A ⁺ RNA was prepared from the obtained total RNA using an mRNA preparation kit (mRNA Purification Kit (Pharmacia)). Then 4 μg of poly A ⁺
CDNA Synthesis Kit (CDNA Synthesis Kit (P
harmacia)) was used to synthesize cDNA.

The obtained cDNA was inserted into a λZAPII vector (Stratagene) and subjected to in vitro packaging to prepare a cDNA library using λ phage.

One clone T6-1 isolated by the differential display method of the above <1> was treated with ³² P.
The 80,000 plaques of the above cDNA library were screened by the plaque hybridization method to obtain positive clones. After hybridization at 60 ° C. for 16 hours, 0.2 × SSC
/ Washed in 0.1% SDS solution at 60 ° C for 10 minutes.
The longest clone among the obtained clones was designated as T6-1-
It was set to 20, and further analysis was performed.

<3> Determination of nucleotide sequence The nucleotide sequence of T6-1-20 was analyzed using a nucleotide sequence determination kit (Bc
When determined by the dideoxy method using aBEST Dideoxy Sequence Kit (manufactured by Takara Shuzo Co., Ltd.), this cDNA consisted of 3,528 bp, and one open reading frame encoding 977 amino acids was found. Then, when a database search was carried out for the amino acid sequence encoded by this cDNA, cysteines that bind to X-box, which are conserved in the promoter region of the human MHC class II gene, and the portion from 1 to 771 of this amino acid sequence. -Rich DNA binding protein (Cystei
39.7% between the amino acid sequence of n-rich DNA-binding protein (NF-X1)) from 343 to 1087
It turned out that there is a homology of.

This NF-X1 has been shown to function as a repressor that regulates the expression of MHC class II genes, and has a new cysteine-rich seven-fold repeating structure in the central part of the gene. It is reported as being a large family. The soybean gene isolated this time is
It has a 7-fold repeating structure rich in cysteine (Fig. 1), and was shown to encode a cysteine-rich DNA binding protein. The gene from which this cDNA was derived was designated as SJIP-2.

<4> Expression analysis of SJIP-2 In order to investigate the expression pattern of the gene (SJIP-2) encoding the cysteine-rich DNA binding protein, R was determined from soybean culture cells treated with jasmonic acid and untreated cells.
NA was extracted, and hybridization was performed using T6-1 (200 bp) as a probe.

RNA was extracted by the guanidine thiocyanate / phenol chloroform method. 1.6m
l 4.23 M guanidine thiocyanate, 25 mM sodium citrate solution, 0.2 ml 20% sarcosyl solution, 0.2 ml 2-mercaptoethanol solution and 2 ml phenol / chloroform solution were mixed and mixed solution About 1.5 g of cultured cells was added to and the cultured cells were crushed. This was centrifuged at 3,000 rpm for 10 minutes, the supernatant was extracted with phenol / chloroform, and then with chloroform. Ethanol was added to the obtained solution to precipitate RNA, and then the precipitate was mixed with 1
It was dissolved in ml of water, 250 μl of 10 M LiCl solution was added, and the mixture was left standing at 4 ° C. for 2 hours or more. Centrifuge this and RN
The A precipitate was collected.

20 μl of RNA obtained as described above
After electrophoresing g on an agarose gel, the RNA was transferred to a nylon membrane. Northern hybridization was carried out using T6-1 labeled with ³² P as a probe.
Hybridization was carried out at 60 ° C. for 16 hours, and then 0.
After washing in a 2 × SSC / 0.1% SDS solution at 60 ° C. for 30 minutes, an autoradiogram was taken using an X-ray film.

As a result, it was revealed that SJIP-2 was not expressed in the cells not treated with jasmonic acid, but was specifically expressed in the cells treated with jasmonic acid.
Therefore, it was revealed that this SJIP-2 is a gene of a DNA binding protein specifically induced by jasmonic acid.

[0045]

INDUSTRIAL APPLICABILITY The DNA of the present invention encodes a jasmonic acid-induced DNA binding protein. This DNA
Binding proteins are believed to be factors that regulate jasmonic acid-induced gene expression. Therefore,
It is expected that it will be possible to control the expression of these genes and be used for the production of plants resistant to pathogenic bacteria, production of useful secondary metabolites, suppression of production of toxic secondary metabolites, etc. .

[0046]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3528 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA Origin organism name: Soybean Strain name: SB-P Sequence features Characteristic symbols : CDS existing position: 450..3480 method to determine the characteristics: P sequence TCACACAGCG CAACTTTCGT CTTCTATCCC CAACCCTAAC CAGGTATCAT CTTCCTATCT 60 TTCCTAAATT TCAGTCAGTC AGAATATTTG TCACTTAATT TGGAATTTTC ATTTCGAGAG 120 TTGAGACTTA GACATGTTAA TTTGACTTTT AGGGTTGAGG TTATCATCAA TCTTTCCATT 180 TTCAATCTGA TGAATTGGAT TTGAATTTTC ATGTGTCAGT TAGCGAACGA TCGATTAGAG 240 TTTCGACGCA GGATGAGTTC GAAAGAGCGA AGCAGCAGCC GCATTCGAGG GTTCCTCGTC 300 GTCAGGAGTG GATTCGTAGA GATGTTGGTG GTTGCTCCAA TCCAAGAAAG CCCAAGAAGG 360 GATCATCTTC GAATTCGAGG GAGGAGTCTA ATTTGCCTCA GC TGC TGC GTC TCT TAT GAC AGT CTC TCT TGC GCT ATC TCT ATC TTT CAC 521 Arg Ser Ala Pro Ile Trp Ser Cys Ser Gly Cys Phe S er Ile Phe His 10 15 20 CTC ACT TGT ATC AAG AAG TGG GCT CGT GCA CCC ATT TCT GTG GAT TTG 569 Leu Thr Cys Ile Lys Lys Trp Ala Arg Ala Pro Ile Ser Val Asp Leu 25 30 35 40 TCC GTT GAG AAG AAC CAG GGC GGC TTC AAT TGG CGT TGC CCT GGT TGC 617 Ser Val Glu Lys Asn Gln Gly Gly Phe Asn Trp Arg Cys Pro Gly Cys 45 50 55 CAG TCT GTG CAG CTC ACT TCA TCC AAG GAT ATT AGG TAT CTA TGC TTC 665 Gln Ser Val Gln Leu Thr Ser Ser Lys Asp Ile Arg Tyr Leu Cys Phe 60 65 70 TGT GGA AAG AGG CCA GAT CCA CCC TCT GAT TTG TAT CTC ATG CCA CAT 713 Cys Gly Lys Arg Pro Asp Pro Pro Ser Asp Leu Tyr Leu Met Pro His 75 80 85 TCC TGT GGA GAA CCA TGT GGC AAG CCT CTT GAG AGG GAC CTT CAA GGG 761 Ser Cys Gly Glu Pro Cys Gly Lys Pro Leu Glu Arg Asp Leu Gln Gly 90 95 100 GAT AAG GAG CTT CTT TGC CCT CAT CTT TGT GTC TTG CAA TGC CAT CCC 809 Asp Lys Glu Leu Leu Cys Pro His Leu Cys Val Leu Gln Cys His Pro 105 110 115 120 GGC CCC TGT CCT CCT TGC AAA GCA TTT GCC CCT CCA CGT CTG TGT CCT 857 Gly Pro Cys Pro Pro Cys Lys Ala Phe Ala Pro Pro Arg Leu Cys Pro 125 130 135 TGT GGG AAG AAA AAT ATT ACC ACT CGT TGC TCT GAC CGC CAG TCT GTT 905 Cys Gly Lys Lys Asn Ile Thr Thr Arg Cys Ser Asp Arg Gln Ser Val 140 145 150 CTT ACC TGT GGC CAG CGC TGC CAA AAG CTT CTT CAA TGT GGC CGT CAT 953 Leu Thr Cys Gly Gln Arg Cys Gln Lys Leu Leu Gln Cys Gly Arg His 155 160 165 CGC TGT CAG CAA ATC TGT CAT CTG GGT CCT TGT CAT CCT TGT CAA GTT 1001 Arg Cys Gln Gln Ile Cys His Leu Gly Pro Cys His Pro Cys Gln Val 170 175 180 CCA ATC AAT GCC TCT TGC TTT TGT GCC CAA AAG ATG GAG GTA ATT CTT 1049 Pro Ile Asn Ala Ser Cys Phe Cys Ala Gln Lys Met Glu Val Ile Leu 185 190 195 200 TGT GGG GAG ATG GCT GTC AAG GGT GAA ATC AGA GCA GAT GGT GGA GTA 1097 Cys Gly Glu Met Ala Val Lys Gly Glu Ile Arg Ala Asp Gly Gly Val 205 210 215 TTC TCT TGT GGT TCC ACT TGT CAA AAG AAA CTT AAT TGT GGT AAT CAT 1145 Phe Ser Cys Gly Ser Thr Cys Gln Lys Lys Leu Asn Cys Gly Asn His 220 225 230 ATC TGT ATC GAG ACT TGT CAT CCA GGT AGC TGT GGG GAC TGT GAA TTA 1193 Ile Cys Ile Glu Thr Cys His Pro Gl y Ser Cys Gly Asp Cys Glu Leu 235 240 245 TTA CCA TCC CGT ATT AAG ACA TGC TGT TGT GGG AAA ACT AGA TTG GAG 1241 Leu Pro Ser Arg Ile Lys Thr Cys Cys Cys Gly Lys Thr Arg Leu Glu 250 255 260 GAG AAA CGC CAC AGT TGT TTA GAC CCA ATT CCT ACC TGT TCA CAA GTA 1289 Glu Lys Arg His Ser Cys Leu Asp Pro Ile Pro Thr Cys Ser Gln Val 265 270 275 280 TGT GGC AAG TAC CTT CCT TGC GGG ATT CAT CAT TGT GAA GAG CCA TGC 1337 Cys Gly Lys Tyr Leu Pro Cys Gly Ile His His Cys Glu Glu Pro Cys 285 290 295 CAT GCT GGG GAT TGT TCT CCT TGT CTG GTT CTA GTT TCT CAG AAG TGT 1385 His Ala Gly Asp Cys Ser Pro Cys Leu Val Leu Val Ser Gln Lys Cys 300 305 310 AGA TGT GGC TCG ACT TCC CGA ACT GTG GAG TGT TGC AAG ACA AAA ATG 1433 Arg Cys Gly Ser Thr Ser Arg Thr Val Glu Cys Cys Lys Thr Lys Met 315 320 325 GAA AAT GAG AAA TTT ACT TGT GAA AGG CCT TGT GGG CAG AAA AAG AAT 1481 Glu Asn Glu Lys Phe Thr Cys Glu Arg Pro Cys Gly Gln Lys Lys Asn 330 335 340 TGT GGA AGG CAT CGA TGT AGT GAA AGG TGT TGT CCA CTT TCT AAT CCA 1529 Cys Gly Arg His Arg Cys Ser Glu Arg Cys Cys Pro Leu Ser Asn Pro 345 350 355 360 AAT AAT ATT CTA AAT GCA GAT TGG GAT CCA CAC TTC TGT CAA TTG CCG 1577 Asn Asn Ile Leu Asn Ala Asp Trp Asp Pro His Phe Cys Gln Leu Pro 365 370 375 TGT GGA AAG AAG TTA AGG TGT GGG CAG CAT GCA TGT GAA TCC CTG TGC 1625 Cys Gly Lys Lys Leu Arg Cys Gly Gln His Ala Cys Glu Ser Leu Cys 380 385 390 CAC AGT GGT CAT TGT CCA CCT TGT CTT GAA ACT TTT ACT GAT TTG 1673 His Ser Gly His Cys Pro Pro Cys Leu Glu Thr Ile Phe Thr Asp Leu 395 400 405 ACA TGT GCT TGT GGT AAG ACT TCA ATC CCT CCT CCA TTG CCT TGT GGC 1721 Thr Cys Ala Cys Gly Lys Thr Ser Ile Pro Pro Pro Leu Pro Cys Gly 410 415 420 ACA CCG CCT CCC TCA TGT CAG CTT CCA TGT TCA GTT CCT CAG CCT TGT 1769 Thr Pro Pro Pro Cys Gln Leu Pro Cys Ser Val Pro Gln Pro Cys 425 430 435 440 TCG CAT CCA GCC TCT CAC AGC TGT CAT TTT GGA GAT TGC CCT CCT TGT 1817 Ser His Pro Ala Ser His Ser Cys His Phe Gly Asp Cys Pro Pro Cys 445 450 455 TCA ATG CCC ATA GCA AAA GAA TGT ATT GGT GGA CAT GTA GTT CTT AGG 1865 Ser Met Pro Ile Ala Lys Glu Cys Ile Gly Gly His Val Val Leu Arg 460 465 470 AAC ATA CCT TGT GGT TCG AAG GAT ATT AAA TGC AAT AAA CTC TGT GGG 1913 Asn Ile Pro Cys Gly Ser Lys Asp Ile Lys Cys Asn Lys Leu Cys Gly 475 480 485 AAG ACC AGA CAG TGT GGT TTA CAT GCA TGT GGC AGA ACA TGT CAC CTC 1961 Lys Thr Arg Gln Cys Gly Leu His Ala Cys Gly Arg Thr Cys His Leu 490 495 500 CCC CCT TGT GAT AAT CTG TCA GCT GTG CCA GGT ATC CGA GCC TCT TGT 2009 Pro Pro Cys Asp Asn Leu Ser Ala Val Pro Gly Ile Arg Ala Ser Cys 505 510 515 520 GGG CAA ACA TGT GGT GCT CCT AGG AGA GAC TGC CGG CAT ACA TGT ACA 2057 Gly Gln Thr Cys Gly Ala Pro Arg Arg Asp Cys Arg His Thr Cys Thr 525 530 535 GCT CCT TGT CAC CCT TCA ACT CCA TGT CCA GAT ACA AGA TGC AAA TTC 2105 Ala Pro Cys His Pro Ser Thr Pro Cys Pro Asp Thr Arg Cys Lys Phe 540 545 550 CCT GTC ACA ATT ACT TGT TCT TGT GGC CGA ATA ACA GAA AAT GTT CCT 2153 Pro Val Thr Ile Thr Cys Ser Cys Gly Arg Ile Thr Glu Asn Val Pro 555 560 565 TGT GAT GCT GGT GGC AGT TGT GCT AAT TAT G AT GCT GAT ACT GTA CAT 2201 Cys Asp Ala Gly Gly Ser Cys Ala Asn Tyr Asp Ala Asp Thr Val His 570 575 580 GAA GCT TCC ATT ATT CAA AAG TTG CCT GTG CTT CTT CAA CCC GTG GCT 2249 Glu Ala Ser Ile Ile Gln Lys Leu Pro Val Leu Leu Gln Pro Val Ala 585 590 595 600 GCA AAT GGC AAA AAA GTC CCC CTC GGA CAA AGA AAA CTG ATG TGT AAT 2297 Ala Asn Gly Lys Lys Val Pro Leu Gly Gln Arg Lys Leu Met Cys Asn 605 610 615 GAT GAC TGT GCT AAG TTA GAG CGG AAA AGG GTT CTT GCA GAT GCT TTT 2345 Asp Asp Cys Ala Lys Leu Glu Arg Lys Arg Val Leu Ala Asp Ala Phe 620 625 630 GAG ATT ACC GCT CCA AAT CTG GAT TCA CTC CAT TTT GGT GAG AAT TCG 2393 Glu Ile Thr Ala Pro Asn Leu Asp Ser Leu His Phe Gly Glu Asn Ser 635 640 645 GTT GCT TCT GAA TTG CTG GCT GAC ATG TTG AGA CGT GAT TCT AAA TGG 2441 Val Ala Ser Glu Leu Leu Ala Asp Met Leu Arg Arg Asp Ser Lys Trp 650 655 660 GTT TTA TCT GTT GAA GAG AGA TGC AAG TTT TTA GTA CTT GGC AAG AGC 2489 Val Leu Ser Val Glu Glu Arg Cys Lys Phe Leu Val Leu Gly Lys Ser 665 670 675 680 AGA GGA AAT GCA C AT GGT CCA AAA GTC CAT GTT TTC TGT CCT ATG TTA 2537 Arg Gly Asn Ala His Gly Pro Lys Val His Val Phe Cys Pro Met Leu 685 690 695 AAG GAC AAA AGA GAT GCA GTG AGG GTG ATT GCT GAG AGA TGG AAG CTT 2585 Lys Asp Lys Arg Asp Ala Val Arg Val Ile Ala Glu Arg Trp Lys Leu 700 705 710 GCA GTG AAT GCA GCT GGT CGG GAG CCA AAG CAT TTC GTA GTT GTT CAT 2633 Ala Val Asn Ala Ala Gly Arg Glu Pro Lys His Phe Val Val Val His 715 720 725 GTT ACA CCA AAA TCA AGA GCT CCT GCT CGT GTG CTA GGG TTT AAG GGT 2681 Val Thr Pro Lys Ser Arg Ala Pro Ala Arg Val Leu Gly Phe Lys Gly 730 735 740 ACT ACA ACT GTA AAT GTA CCC CTT CCT CCG GCA TTT GAT CCT TTG GTT 2729 Thr Thr Thr Val Asn Val Pro Leu Pro Pro Ala Phe Asp Pro Leu Val 745 750 755 760 GAT ATG GAT CCT CGA CTT GTT GTC TCT TTT ATA GAC TTA CCA ATG GAT 2777 Asp Met Asp Pro Arg Leu Val Val Ser Phe Ile Asp Leu Pro Met Asp 765 770 775 GCA GAT ATT AGT GCA TTG GTG TTG AGA TTT GGT GGT GAG TGT GAA CTT 2825 Ala Asp Ile Ser Ala Leu Val Leu Arg Phe Gly Gly Glu Cys Glu Leu 780 785 79 0 GTT TGG TTA AAT GAC AAA AAT GCA TTG GCC GTT TTT AAT GAC CCT GCC 2873 Val Trp Leu Asn Asp Lys Asn Ala Leu Ala Val Phe Asn Asp Pro Ala 795 800 805 CGT GCT GCA ACT GCA ATG AGG AGG TTG GAT CAT GGT ACG GTT TAT CAG 2921 Arg Ala Ala Thr Ala Met Arg Arg Leu Asp His Gly Thr Val Tyr Gln 810 815 820 GGA GCT GTG GTG GTG GTT GTT CCA AAT GTC GGG GCA TCA GTA GCA TCT 2969 Gly Ala Val Val Val Val Val Pro Asn Val Gly Ala Ser Val Ala Ser 825 830 835 840 TCA GCT ACC AAT GCC TGG GGA GGA TCT GGG ACA ATG AAA GGA GGA GCA 3017 Ser Ala Thr Asn Ala Trp Gly Gly Ser Gly Thr Met Lys Gly Gly Ala 845 850 855 CTG GCA GCA TTA AAG AGT AAT CCA TGG AAA AAG GAT GTT ATT CAA GAG 3065 Leu Ala Ala Leu Lys Ser Asn Pro Trp Lys Lys Asp Val Ile Gln Glu 860 865 870 CCA GGT TGG AGA GAA GAT GCT TGG GGT GAT GAG GAG TGG GCT ACT GGT 3113 Pro Gly Trp Arg Glu Asp Ala Trp Gly Asp Glu Glu Trp Ala Thr Gly 875 880 885 TCT GCT AAT GTC AAA TTG CCT ATT CAG AAG AAA GAA GCC CGA ATA TCT 3161 Ser Ala Asn Val Lys Leu Pro Ile Gln Lys Lys Glu Ala Arg Ile Ser 890 895 900 GCT TCA GTA AAT CCT TGG AGT GTC CTA AAT CAA GAA TCG TCT TCA AGT 3209 Ala Ser Val Asn Pro Trp Ser Val Leu Asn Gln Glu Ser Ser Ser Ser 905 910 915 920 TCA TCT GTT GCA GCC ATT AAA ATT GAT GGT TCT AGG AAA CAC TCT GAA 3257 Ser Ser Val Ala Ala Ile Lys Ile Asp Gly Ser Arg Lys His Ser Glu 925 930 935 AGT AGT GTT ATC ACA AAG TTG GAG CCT CGT GAT GGT GGT TCA AAT CTA 3305 Ser Ser Val Ile Thr Lys Leu Glu Pro Arg Asp Gly Gly Ser Asn Leu 940 945 950 GGA GGG CAG CCT GCA GGA AAC TTT GAT GCT TTG GAA GCT TCT GAT GTA 3353 Gly Gly Gln Pro Ala Gly Asn Phe Asp Ala Leu Glu Ala Ser Asp Val 955 960 965 GTA GAT GAT TGG GAG AAG GCT TGT GAA TAGCAAGGAG TAAACATTTT 3400 Val Asp Asp Trp Glu Lys Ala Cys Glu 970 975 TCATTTTATT TTGTTGAGAA GGAGGCAGAT TTTGACTAGT GAAATGTCTACATCCCTACATATTATTTATAGCTCTTTT 3460 TGTCCACTAGATGTATTTTA TGCTT

SEQ ID NO: 2 Sequence length: 977 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ile Cys Tyr Asp Met Val Arg Arg Ser Ala Pro Ile Trp Ser Cys 1 5 10 15 Ser Gly Cys Phe Ser Ile Phe His Leu Thr Cys Ile Lys Lys Trp Ala 20 25 30 Arg Ala Pro Ile Ser Val Asp Leu Ser Val Glu Lys Asn Gln Gly Gly 35 40 45 Phe Asn Trp Arg Cys Pro Gly Cys Gln Ser Val Gln Leu Thr Ser Ser 50 55 60 Lys Asp Ile Arg Tyr Leu Cys Phe Cys Gly Lys Arg Pro Asp Pro Pro 65 70 75 80 Ser Asp Leu Tyr Leu Met Pro His Ser Cys Gly Glu Pro Cys Gly Lys 85 90 95 Pro Leu Glu Arg Asp Leu Gln Gly Asp Lys Glu Leu Leu Cys Pro His 100 105 110 Leu Cys Val Leu Gln Cys His Pro Gly Pro Cys Pro Pro Cys Lys Ala 115 120 125 Phe Ala Pro Pro Arg Leu Cys Pro Cys Gly Lys Lys Asn Ile Thr Thr 130 135 140 Arg Cys Ser Asp Arg Gln Ser Val Leu Thr Cys Gly Gln Arg Cys Gln 145 150 155 160 Lys Leu Leu Gln Cys Gly Arg His Arg Cys Gln Gln Ile Cys His Leu 165 170 175 Gly Pro Cys His Pr o Cys Gln Val Pro Ile Asn Ala Ser Cys Phe Cys 180 185 190 Ala Gln Lys Met Glu Val Ile Leu Cys Gly Glu Met Ala Val Lys Gly 195 200 205 Glu Ile Arg Ala Asp Gly Gly Val Phe Ser Cys Gly Ser Thr Cys Gln 210 215 220 Lys Lys Leu Asn Cys Gly Asn His Ile Cys Ile Glu Thr Cys His Pro 225 230 235 240 Gly Ser Cys Gly Asp Cys Glu Leu Leu Pro Ser Arg Ile Lys Thr Cys 245 250 255 Cys Cys Gly Lys Thr Arg Leu Glu Glu Lys Arg His Ser Cys Leu Asp 260 265 270 Pro Ile Pro Thr Cys Ser Gln Val Cys Gly Lys Tyr Leu Pro Cys Gly 275 280 285 Ile His His Cys Glu Glu Pro Cys His Ala Gly Asp Cys Ser Pro Cys 290 295 300 Leu Val Leu Val Ser Gln Lys Cys Arg Cys Gly Ser Thr Ser Arg Thr 305 310 315 320 Val Glu Cys Cys Lys Thr Lys Met Glu Asn Glu Lys Phe Thr Cys Glu 325 330 335 Arg Pro Cys Gly Gln Lys Lys Asn Cys Gly Arg His Arg Cys Ser Glu 340 345 350 Arg Cys Cys Pro Leu Ser Asn Pro Asn Asn Ile Leu Asn Ala Asp Trp 355 360 365 Asp Pro His Phe Cys Gln Leu Pro Cys Gly Lys Lys Leu Arg Cys Gly 370 375 380 Gln His Ala Cys Glu S er Leu Cys His Ser Gly His Cys Pro Pro Cys 385 390 395 400 Leu Glu Thr Ile Phe Thr Asp Leu Thr Cys Ala Cys Gly Lys Thr Ser 405 410 415 Ile Pro Pro Pro Leu Pro Cys Gly Thr Pro Pro Ser Sers Gln Leu 420 425 430 Pro Cys Ser Val Pro Gln Pro Cys Ser His Pro Ala Ser His Ser Cys 435 440 445 His Phe Gly Asp Cys Pro Pro Cys Ser Met Pro Ile Ala Lys Glu Cys 450 455 460 Ile Gly Gly His Val Val Leu Arg Asn Ile Pro Cys Gly Ser Lys Asp 465 470 475 480 Ile Lys Cys Asn Lys Leu Cys Gly Lys Thr Arg Gln Cys Gly Leu His 485 490 495 Ala Cys Gly Arg Thr Cys His Leu Pro Pro Cys Asp Asn Leu Ser Ala 500 505 510 Val Pro Gly Ile Arg Ala Ser Cys Gly Gln Thr Cys Gly Ala Pro Arg 515 520 525 Arg Asp Cys Arg His Thr Cys Thr Ala Pro Cys His Pro Ser Thr Pro 530 535 540 Cys Pro Asp Thr Arg Cys Lys Phe Pro Val Thr Ile Thr Cys Ser Cys 545 550 555 560 565 570 575 Asn Tyr Asp Ala Asp Thr Val His Glu Ala Ser Ile Ile Gln Lys Leu 580 585 590 Pro Val Leu Leu Gln Pro Val Ala Ala Asn Gly Lys Lys Val Pro Leu 595 600 605 Gly Gln A rg Lys Leu Met Cys Asn Asp Asp Cys Ala Lys Leu Glu Arg 610 615 620 Lys Arg Val Leu Ala Asp Ala Phe Glu Ile Thr Ala Pro Asn Leu Asp 625 630 635 640 Ser Leu His Phe Gly Glu Asn Ser Val Ala Ser Glu Leu Leu Ala Asp 645 650 655 Met Leu Arg Arg Asp Ser Lys Trp Val Leu Ser Val Glu Glu Arg Cys 660 665 670 Lys Phe Leu Val Leu Gly Lys Ser Arg Gly Asn Ala His Gly Pro Lys 675 680 685 Val His Val Phe Cys Pro Met Leu Lys Asp Lys Arg Asp Ala Val Arg 690 695 700 Val Ile Ala Glu Arg Trp Lys Leu Ala Val Asn Ala Ala Gly Arg Glu 705 710 715 720 Pro Lys His Phe Val Val Val His Val Thr Pro Lys Ser Arg Ala Pro 725 730 735 Ala Arg Val Leu Gly Phe Lys Gly Thr Thr Thr Val Asn Val Pro Leu 740 745 750 Pro Pro Ala Phe Asp Pro Leu Val Asp Met Asp Pro Arg Leu Val Val 755 760 765 Ser Phe Ile Asp Leu Pro Met Asp Ala Asp Ile Ser Ala Leu Val Leu 770 775 780 Arg Phe Gly Gly Glu Cys Glu Leu Val Trp Leu Asn Asp Lys Asn Ala 785 790 795 800 Leu Ala Val Phe Asn Asp Pro Ala Arg Ala Ala Thr Ala Met Arg Arg 805 810 815 Leu AspHis Gly Thr Val Tyr Gln Gly Ala Val Val Val Val Val Pro 820 825 830 Asn Val Gly Ala Ser Val Ala Ser Ser Ala Thr Asn Ala Trp Gly Gly 835 840 845 Ser Gly Thr Met Lys Gly Gly Ala Leu Ala Ala Leu Lys Ser Asn Pro 850 855 860 Trp Lys Lys Asp Val Ile Gln Glu Pro Gly Trp Arg Glu Asp Ala Trp 865 870 875 880 Gly Asp Glu Glu Trp Ala Thr Gly Ser Ala Asn Val Lys Leu Pro Ile 885 890 895 Gln Lys Lys Glu Ala Arg Ile Ser Ala Ser Val Asn Pro Trp Ser Val 900 905 910 Leu Asn Gln Glu Ser Ser Ser Ser Ser Ser Val Ala Ala Ile Lys Ile 915 920 925 Asp Gly Ser Arg Lys His Ser Glu Ser Ser Val Ile Thr Lys Leu Glu 930 935 940 Pro Arg Asp Gly Gly Ser Asn Leu Gly Gly Gln Pro Ala Gly Asn Phe 945 950 955 960 Asp Ala Leu Glu Ala Ser Asp Val Val Asp Asp Trp Glu Lys Ala Cys 965 970 975 Glu

SEQ ID NO: 3 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other DNA. . Synthetic DNA sequence TTTTTTTTTT TTVG 14

SEQ ID NO: 4 Sequence length: 14 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other DNA. . Synthetic DNA sequence TTTTTTTTTT TTVA 14

SEQ ID NO: 5 Sequence length: 14 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other DNA. . Synthetic DNA sequence TTTTTTTTTT TTVT 14

SEQ ID NO: 6 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other DNA. . Synthetic DNA sequence TTTTTTTTTT TTVC 14

[0052]

[Brief description of drawings]

FIG. 1 is a diagram showing a cysteine-rich 7-fold repeating structure possessed by a cDNA (NF-X1) encoding the DNA binding protein of the present invention. The numbers on the left and right of each column represent the amino acid number from the N-terminus.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12Q 1/68 C12N 5/00 C (72) Inventor Hiroyuki Ota 4259 Nagatsuda, Midori-ku, Yokohama Faculty of Life Science and Engineering, Institute of Technology

Claims (6)

[Claims] 1. Having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, it may have one or more amino acid substitutions, deletions, insertions, additions or transitions as long as the DNA binding activity is not substantially impaired. DNA encoding a DNA binding protein. 2. The DNA according to claim 1, which has a nucleotide sequence represented by nucleotide numbers 450 to 3480 in the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing. 3. A DNA having at least a part of a base sequence complementary to the base sequence shown in SEQ ID NO: 1. 4. A plant transformed with the DNA of claim 1. 5. A plant transformed with the DNA according to claim 3. 6. The amino acid sequence having the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing, which may have one or more amino acid substitutions, deletions, insertions, additions or transitions as long as it does not substantially impair the DNA binding activity. DNA binding protein.